Sciatic Ligation Model In Rats

Brief Description

Assessment of the in vivo effect of an investigational compound on the reduction in neuropathic pain following sciatic ligation in rats.

Introduction

Many of the broad-spectrum ASDs are effective in modulating abnormal neuronal activity in a number of CNS disorders in addition to epilepsy, including bipolar disorder, migraine, and neuropathic pain [1]. Often, these comorbidities of epilepsy can be more detrimental to a patient’s quality of life than the seizures themselves [2]. The 2012 Institute of Medicine’s report acknowledges several painful somatic disorders, such as fibromyalgia and osteoarthritis, as well as painful neurological conditions, such as migraine, chronic pain syndromes, and neuropathic pain, as comorbid conditions associated with epilepsy [3, 4]. Thus, one approach towards finding therapeutics with efficacy for these comorbidities associated with epilepsy is to expand the preclinical screening of the effects of an investigational compound to include models of nociception and neuropathic pain. Like epilepsy itself, neuropathic pain represent pathological states of abnormal neuronal discharges [5]. At the molecular level, most, if not all, of the available ASDs target receptor- and voltage-gated ion channels that contribute to abnormal neuronal discharges. Thus, it is not necessarily unexpected that those ASDs with broad neuromodulatory activities would be clinically useful for the treatment of neurological and somatic disorders associated with acute, inflammatory, or neuropathic pain. The adaptive responses that take place in the peripheral nervous system and spinal cord following an acute injury lead to a hyper-responsiveness to a non-noxious stimulation, known as allodynia [5]. In an attempt to broaden the therapeutic application of a novel neuromodulatory compound identified in the traditional seizure models, the ETSP has examined the efficacy of the most promising and/or novel investigational ASDs in several models of nociception, including the partial sciatic nerve ligation model of chronic allodynia in rats (Test 23) and the formalin test model of hyperalgesia in mice (Test 22).

Methods

Partial sciatic nerve ligation surgery is performed according to the methods described by Seltzer [6]. Just prior to surgery, rats receive 0.1 mg/kg buprenorphine, s.c., for management of surgical and post-operative pain. General anesthesia is induced with 2% isoflurane and maintained throughout the ligation procedure at 1-1.5% in compressed air. The depth of anesthesia is confirmed by carefully monitoring the depth of respiration and the lack of response to tail pinch. The level of isoflurane is adjusted throughout the procedure as needed to prevent any unnecessary pain or distress. Rats are sterilely prepared for surgical manipulations, and the underlying muscle of the upper thigh is then separated to expose the sciatic nerve and approximately 1/3 to 1/2 of the nerve is tied off by passing a "tapered 130-4" needle attached to size 7 nylon suture through the distal sciatic nerve. Importantly, to maintain motor function and to prevent the animal from chewing on the ligated limb, no more than half the distal sciatic nerve is ligated. The incisions are sutured and animals are then allowed to recover from the anesthetic. Twelve hours post-surgery, rats receive a second dose of buprenorphine to minimize discomfort. The rats are closely monitored for 7 days for the development of infection or other adverse outcomes. Following a 7-10 day post-operative recovery period, the rats are tested for the development of consistent, mechanical allodynia (pain response to a non-noxious stimulus) in the ipsilateral (ligated) versus the contralateral (non-ligated) leg. Briefly, rats are individually placed in a bottomless plexiglass box atop a 1/4" stainless (or galvanized) steel platform. After a 30-min habituation period, the baseline mechanical sensitivity of each rat is determined by applying a series of calibrated von Frey monofilaments perpendicularly to the plantar surface of each hind paw in between the pads or further back toward the heel [7]. The probe is applied with sufficient pressure to bend the fiber and then held in place for up to 6 sec. The 50% threshold for foot withdrawal is determined by using the “Up and Down” step procedure [7], such that, after a positive response (withdrawal of the foot) is noted, a weaker fiber is applied. If there is no spontaneous recoil, or flinch, the next larger diameter fiber is applied, and so forth. This is repeated for 5 steps during the course of the study. However, if the pressure of the fiber raises the foot, it is considered as “no response”. On the testing day, a group of 8 rats, previously determined to have developed consistent mechanical allodynia, are initially screened for the pre-drug baseline sensitivity level to mechanical pain. Following the baseline screening, rats are then administered the investigational compound at a dose approximating the MES ED50 (i.p., as determined in Test 10) and the mechanical threshold to pain is measured 0.5, 1, 2, 4, and 6 hr post-drug administration (or as otherwise indicated by prior data in other tests or Test 10). The mean threshold (± SEM) for foot withdrawal from each time points are then compared to the initial mean pre-drug baseline sensitivity.

Results

To further examine the effects of antiseizure compounds on neuropathic pain, investigational compounds are tested at doses near the MES ED50 (i.p.) for any effects against mechanical allodynia due to partial sciatic nerve ligation in rats. The average and S.E.M. of the pre-drug withdrawal threshold are calculated. At each time point after drug administration, each animal’s response is compared to its pre-drug response and a percent of pre-drug paw withdrawal threshold (PWT) is determined for each rat using the pre-drug PWT as a control; thus, each animal serves as its own control. The % pre-drug values for all 8 rats are then averaged at each time point to determine the mean % of pre-drug PWT for each time point. This is repeated across all five time points tested (0.5, 1, 2, 4, and 6 hr post-drug administration, or as otherwise indicated by prior data in other tests). The average, S.E.M., and n for each time point are compared to that of the pre-drug PWT by one-way ANOVA with Dunnett post-hoc comparison. The TPE is defined as the time point showing the largest increase in the average % of the pre-drug PWT. All time points with a significant increase in PWT are compared to pre-drug PWT, with p < 0.05 considered statistically significant.

Discussion

Recent work has demonstrated that ASDs can frequently attenuate pain in several animal models of inflammatory and neuropathic pain [8]. For example, gabapentin is often clinically prescribed for neuropathic pain, as well as for epilepsy. One explanation for these observations is that epilepsy and neuropathic pain both arise from pathological states of neuronal hyperexcitability. In epilepsy, such central neuron hyperexcitability manifests in seizures, whereas in neuropathic pain, this peripheral neuron hyperexcitability results in chronic repetitive firing that is perceived as pain [1] and, if not addressed adequately, can result in central sensitization via a process very similar to kindling for chronic seizure presentation [9]. Therefore, the ability of investigational compounds to attenuate the neuropathic pain following partial sciatic nerve ligation in rats has been evaluated at the ETSP. Compounds that, when tested at a dose near the rat MES ED50, show significant increases in the response to mechanical allodynia may be effective for chronic, neuropathic pain. Due to the shared neuronal hyperexcitability pathways underlying epilepsy and chronic pain syndromes, including neuropathic pain, compounds that are effective in a variety of animal seizure models and models of allodynia and hyperalgesia may ultimately find clinically utility in alleviating comorbidities associated with epilepsy, including neuropathic pain.